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Biosafety classification is based on U.S. Public Health Service Guidelines, it is the responsibility of the customer to ensure that their facilities comply with biosafety regulations for their own country.

Harvest cells from a culture which is at or near peak density by centrifugation at ~800 x g for 5 min.

Adjust concentration of cells to 2 x 107/mL in fresh medium. If the concentration is too low, centrifuge at ~800 x g for 5 minutes and resuspend the cell pellet with a volume of supernatant to yield the desired concentration.

While cells are centrifuging, prepare a 20% (v/v) solution of sterile DMSO in fresh medium (broth). The DMSO solution when first prepared will warm up due to chemical heat. The solution should be allowed to return to room temperature prior to use.

Mix the cell preparation and the DMSO solution in equal portions. The final concentration will be 107 cells/mL and 10% (v/v) DMSO. The time from the mixing of the cell preparation and DMSO stock solution before the freezing process is begun should be no less than 15 min and no more than 30 min.

Place the ampules in a Nalgene 1°C freezing apparatus. Place the apparatus at -80°C for 1.5 to 2 hours and then plunge ampules into liquid nitrogen. (The cooling rate in this apparatus is approximately -1°C/min.) If freezing unit can compensate for the heat of fusion, maintain rate at -1°C/min through heat of fusion. At -40°C, plunge ampules into liquid nitrogen.

Store in either the vapor or liquid phase of a nitrogen refrigerator.

To thaw a frozen ampule, place it in a 35°C water bath such that the lip of the ampule remains above the water line. Thawing time is approximately 2 to 3 minutes. Do not agitate the ampule. Do not leave ampule in water bath after it is thawed.

Remove the vial from the water bath immediately after thawing. Aseptically transfer the contents of the ampule into 10 mL of fresh ATCC medium 28.

Customers located in the state of Hawaii will need to contact the Hawaii Department of Agriculture to determine if an Import Permit is required. A copy of the permit or documentation that a permit is not required must be sent to ATCC in advance of shipment.